Proliferation of smooth muscle cells in blood vessel walls occurs in response to vascular injury, and in association with certain disease states (Austin, G. E., et al., J Am Coll Cardiol (1985) 6:369-375). The proliferation of these cells can have negative effects due to the production of excess proteins or other matrix molecules, which, along with the cells themselves, form pathologic lesions of, for example, atherosclerosis, renal hypertension, pulmonary hypertension, vasculitis, and postsurgical vascular retinosis. These results are distinguished from the acute response to trauma characterized by blood clotting.
Glycosaminoglycans (GAG) are alternating copolymers of a hexosamine and an aldouronic acid which are found in sulfated forms and are synthesized as proteoglycans. They have collectively been called mucopolysaccharides. To place the compositions discussed below in context, it may be noted that heparin and heparan sulfate are members of the GAG family which are classified by the nature of the hexosamine/aldouronic acid repeating units. For example, in chondroitin sulfates, the aldouronic acid is primarily D-glucuronic acid, and the hexosamine is acetylated 2-amino-2-deoxy-D-galactose (N-acetyl galactosamine, GalNac). In dermatan sulfate (chondroitin sulfate B) the aldouronic acid is mostly L-iduronic acid and the hexosamine is GalNAc. In keratin sulfate, the aldouronic acid is replaced by D-galactose, and the hexosamine is mostly acetylated 2-amino-2-deoxy-D-glucose (N-acetyl glucosamine, GlcNAc). In the compositions of interest herein, heparan sulfate and heparin, the hexosamine is mostly acetylated and sulfated glucosamine (GlcNH.sub.2), and the aldouronic acid is mostly L-iduronic in heparin and mostly D-glucuronic acid in heparan sulfate. Heparan sulfate is commonly considered to have a higher proportion of glucuronic acid than heparin.
Problems of heterogeneity in preparations of heparan sulfate or heparin isolated from tissues make sharp distinctions difficult, since these oligosaccharides are related by the biosynthesis pathway, as explained below. Conventional heparin (used as an anti-coagulant) has a molecular weight of 5-25 kd and is extracted as a mixture of various chain lengths by conventional procedures. These procedures involve autolysis and extraction of suitable tissues, such as beef or porcine lung, intestine, or liver, and removal of nonpolysaccharide components.
The molecular weight of the chains in the extract is significantly lower than the 60-100 kd known to exist in the polysaccharide chains of the heparin proteoglycan synthesized in the tissue. The GAG moiety is synthesized bound to a peptide matrix at a serine residue through a tetrasaccharide linkage region of the sequence Xyl-Gal-Gal-D-GlcA-, which is then elongated at the xylose residue with alternate additions of GlcNac and D-glucuronic acid. The polysaccharide sidechains are modified by a series of enzymes which sequentially deacetylate the N-acetyl glucosamine and replace the acetyl group with sulfate, epimerize the hydroxyl at C5 of the D-glucuronic acid residue (to convert it to L-iduronic acid and the GAG chain from the heparan type to a heparin type), sulfate the O-2 of the resulting L-iduronic acid and then sulfate the O-6 of the glucosamine residue. Some of the chains are further sulfated at the O-3 of the glucosamine residue, either at the heparan or heparin stage. This further sulfation is associated with the active site for antithrombin (anticlotting) activity. Other chemically possible sulfation sites are on the O-3 of L-iduronic or D-glucuronic and O-2 of D-glucuronic acid; however, these are seldom found.
Due to their obvious chemical similarity, isolated "heparin" may contain considerable amounts of what might otherwise be classified as heparan sulfate.
There is an extensive art concerning depolymerization of heparin/heparan sulfate chains and separation of products by size. Particularly relevent is the report of Guo, Y. et al., Anal Biochem (1988) 168:54-62 which discloses the results of structure determination after the 2,5-anhydromannose at the reducing terminus is reduced to the corresponding 2,5-anhydromannitol.
The following tetrasaccharides were listed specifically by Guo. In these representations, the following abbreviations are used: D-glucuronic acid=GlcA; L-iduronic acid=IdoA; D-glucosamine=GlcNH.sub.2 ; N-acetyl-D-glucosamine=GlcNAc; D-glucosamine N-sulfate=GlcNS; 2,5-anhydromannose=Man(2,5); 2,5-anhydromannitol=ManH(2,5). The location of the O-linked sulfate residues is indicated by "S" and the number of the position of sulfation where the SO.sub.3 R residue is linked to oxygen. In the designations below, the alpha and beta anomeric linkages are as those conventionally found in heparin and the indicated D or L configurations as set forth above pertains. The locations of the sulfates are shown below the abbreviation for the sugar to which they apply. ##STR1## (RC represents the ring contracted form analagous to ManH(2,5); it is believed this form is formed when the resulting intermediate hemiacetal is reduced.)
The involvement of heparin or heparan sulfate or degradation products thereof in smooth muscle proliferation has been recognized for some time. Heparin and heparan sulfate can slow or arrest the vascular proliferation associated with injury described hereinabove (Clowes, A. W., et al., Nature (1977) 265:625-626). The effect of heparan sulfate and heparin on smooth muscle proliferation is also described by Marcum, J. A., et al. in Biology of Proteoglycan, Academic Press (1987) pp. 301-343. The inhibition of vascular smooth muscle cell growth by heparin was further described by Castellot, J. J. Jr., al., J Biol Chem (1982) 257:11256-11260 and the effect of heparin on vascular smooth muscle cell growth in fetal tissue was described by Benitz, W. E., et al., J Cell Physiol (1986) 127:1-7. The effect of heparin as an inhibitor of both pericyte and smooth muscle cell proliferation was shown by Orlidge, A., et al., Microvascular Research (1986) 31:41-53, and these authors further showed that chondroitin sulfate, and dermatan sulfate do not have this effect. A review of the effects of heparin and heparan sulfate on the proliferation of smooth muscle cells is in press by Benitz, W. E. in "The Pulmonary Circulation: Normal and Abnormal", Fishman, A. P., ed., University of Pennsylvania Press (1988).
It is not clear by what mechanism these glycosaminoglycans operate, or to what extent they interact with other growth factors such as epithelial and fibroblast growth factors. It has been proposed that a 3-O sulfate on an oligosaccharide of at least 5 sugars is important in this process (Castellot et al., J Cell Biol (1986) 102:1979-1984.
It has now been found that an enhanced antiproliferative activity with respect to smooth muscle cells is associated with a smaller oligosaccharide portion of the heparin or heparan sulfate GAGs.